Fusion-fission-mitophagy cycling and metabolic reprogramming coordinate nerve growth factor (NGF)-dependent neuronal differentiation.

Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion-fission-mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.

Iron and Ferroptosis More than a Suspect: Beyond the Most Common Mechanisms of Neurodegeneration for New Therapeutic Approaches to Cognitive Decline and Dementia.

Neurodegeneration is a multifactorial process that involves multiple mechanisms. Examples of neurodegenerative diseases are Parkinson's disease, multiple sclerosis, Alzheimer's disease, prion diseases such as Creutzfeldt-Jakob's disease, and amyotrophic lateral sclerosis. These are progressive and irreversible pathologies, characterized by neuron vulnerability, loss of structure or function of neurons, and even neuron demise in the brain, leading to clinical, functional, and cognitive dysfunction and movement disorders. However, iron overload can cause neurodegeneration. Dysregulation of iron metabolism associated with cellular damage and oxidative stress is reported as a common event in several neurodegenerative diseases. Uncontrolled oxidation of membrane fatty acids triggers a programmed cell death involving iron, ROS, and ferroptosis, promoting cell death. In Alzheimer's disease, the iron content in the brain is significantly increased in vulnerable regions, resulting in a lack of antioxidant defenses and mitochondrial alterations. Iron interacts with glucose metabolism reciprocally. Overall, iron metabolism and accumulation and ferroptosis play a significant role, particularly in the context of diabetes-induced cognitive decline. Iron chelators improve cognitive performance, meaning that brain iron metabolism control reduces neuronal ferroptosis, promising a novel therapeutic approach to cognitive impairment.

Alzheimer's Disease from the Amyloidogenic Theory to the Puzzling Crossroads between Vascular, Metabolic and Energetic Maladaptive Plasticity.

Alzheimer's disease (AD) is a progressive and degenerative disease producing the most common type of dementia worldwide. The main pathogenetic hypothesis in recent decades has been the well-known amyloidogenic hypothesis based on the involvement of two proteins in AD pathogenesis: amyloid ß (Aß) and tau. Amyloid deposition reported in all AD patients is nowadays considered an independent risk factor for cognitive decline. Vascular damage and blood-brain barrier (BBB) failure in AD is considered a pivotal mechanism for brain injury, with increased deposition of both immunoglobulins and fibrin. Furthermore, BBB dysfunction could be an early sign of cognitive decline and the early stages of clinical AD. Vascular damage generates hypoperfusion and relative hypoxia in areas with high energy demand. Long-term hypoxia and the accumulation within the brain parenchyma of neurotoxic molecules could be seeds of a self-sustaining pathological progression. Cellular dysfunction comprises all the elements of the neurovascular unit (NVU) and neuronal loss, which could be the result of energy failure and mitochondrial impairment. Brain glucose metabolism is compromised, showing a specific region distribution. This energy deficit worsens throughout aging. Mild cognitive impairment has been reported to be associated with a glucose deficit in the entorhinal cortex and in the parietal lobes. The current aim is to understand the complex interactions between amyloid ß (Aß) and tau and elements of the BBB and NVU in the brain. This new approach aimed at the study of metabolic mechanisms and energy insufficiency due to mitochondrial impairment would allow us to define therapies aimed at predicting and slowing down the progression of AD.

Polystyrene micro and nano-particles induce metabolic rewiring in normal human colon cells: A risk factor for human health.

Polystyrene is a thermoplastic polymer widely used in commercial products. Like all plastics, polystyrene can be degraded into microplastic and nanoplastic particles and ingested via food chain contamination. Although the ecological impact due to plastic contamination is well known, there are no studies indicating a carcinogenic potential of polystyrene microplastics (MPs) and nanoplastics (NPs). Here, we evaluated the effects of the MPs and NPs on normal human intestinal CCD-18Co cells. Our results show that internalization of NPs and MPs induces metabolic changes under both acute and chronic exposure by inducing oxidative stress, increasing glycolysis via lactate to sustain energy metabolism and glutamine metabolism to sustain anabolic processes. We also show that this decoupling of nutrients mirrors the effect of the potent carcinogenic agent azoxymethane and HCT15 colon cancer cells, carrying out the typical strategy of cancer cells to optimize nutrients utilization and allowing metabolic adaptation to environmental stress conditions. Taken together our data provide new evidence that chronic NPs and MPs exposure could act as cancer risk factor for human health.

Matrix metalloproteinases, purinergic signaling, and epigenetics: hubs in the spinal neuroglial network following peripheral nerve injury.

Activation of glial cells (reactive gliosis) and the purinergic pathway, together with metalloproteinase (MMP)-induced remodeling of the neural extracellular matrix (nECM), drive maladaptive changes in the spinal cord following peripheral nerve injury (PNI). We evaluated the effects on spinal maladaptive plasticity through administration of oxidized ATP (oxATP), an antagonist of P2X receptors (P2XR), and/or GM6001, an inhibitor of MMPs, in rats following spared nerve injury (SNI) of the sciatic nerve. With morpho-molecular techniques, we demonstrated a reduction in spinal reactive gliosis and changes in the neuro-glial-nECM crosstalk via expression remodeling of P2XR, nerve growth factor (NGF) receptors (TrkA and p75), and histone deacetylase 2 (HDAC2) after treatments with oxATP/GM6001. Altogether, our data suggest that MMPs and purinergic inhibition have a modulatory impact on key proteins in the neuro-glial-nECM network, acting at different levels from intracellular signaling to epigenetic modifications.

The Spatiotemporal Coupling: Regional Energy Failure and Aberrant Proteins in Neurodegenerative Diseases.

The spatial and temporal coordination of each element is a pivotal characteristic of systems, and the central nervous system (CNS) is not an exception. Glial elements and the vascular interface have been considered more recently, together with the extracellular matrix and the immune system. However, the knowledge of the single-element configuration is not sufficient to predict physiological or pathological long-lasting changes. Ionic currents, complex molecular cascades, genomic rearrangement, and the regional energy demand can be different even in neighboring cells of the same phenotype, and their differential expression could explain the region-specific progression of the most studied neurodegenerative diseases. We here reviewed the main nodes and edges of the system, which could be studied to develop a comprehensive knowledge of CNS plasticity from the neurovascular unit to the synaptic cleft. The future goal is to redefine the modeling of synaptic plasticity and achieve a better understanding of neurological diseases, pointing out cellular, subcellular, and molecular components that couple in specific neuroanatomical and functional regions.

Transcriptomics and Metabolomics Integration Reveals Redox-Dependent Metabolic Rewiring in Breast Cancer Cells.

Rewiring glucose metabolism toward aerobic glycolysis provides cancer cells with a rapid generation of pyruvate, ATP, and NADH, while pyruvate oxidation to lactate guarantees refueling of oxidized NAD+ to sustain glycolysis. CtPB2, an NADH-dependent transcriptional co-regulator, has been proposed to work as an NADH sensor, linking metabolism to epigenetic transcriptional reprogramming. By integrating metabolomics and transcriptomics in a triple-negative human breast cancer cell line, we show that genetic and pharmacological down-regulation of CtBP2 strongly reduces cell proliferation by modulating the redox balance, nucleotide synthesis, ROS generation, and scavenging. Our data highlight the critical role of NADH in controlling the oncogene-dependent crosstalk between metabolism and the epigenetically mediated transcriptional program that sustains energetic and anabolic demands in cancer cells.

Inhibition of plasminogen/plasmin system retrieves endogenous nerve growth factor and adaptive spinal synaptic plasticity following peripheral nerve injury.

Dysfunctions of the neuronal-glial crosstalk and/or impaired signaling of neurotrophic factors represent key features of the maladaptive changes in the central nervous system (CNS) in neuroinflammatory as neurodegenerative disorders. Tissue plasminogen activator (tPA)/plasminogen (PA)/plasmin system has been involved in either process of maturation and degradation of nerve growth factor (NGF), highlighting multiple potential targets for new therapeutic strategies. We here investigated the role of intrathecal (i.t.) delivery of neuroserpin (NS), an endogenous inhibitor of plasminogen activators, on neuropathic behavior and maladaptive synaptic plasticity in the rat spinal cord following spared nerve injury (SNI) of the sciatic nerve. We demonstrated that SNI reduced spinal NGF expression, induced spinal reactive gliosis, altering the expression of glial and neuronal glutamate and GABA transporters, reduced glutathione (GSH) levels and is associated to neuropathic behavior. Beside the increase of NGF expression, i.t. NS administration reduced reactive gliosis, restored synaptic homeostasis, GSH levels and reduced neuropathic behavior. Our results hereby highlight the essential role of tPA/PA system in the synaptic homeostasis and mechanisms of maladaptive plasticity, sustaining the beneficial effects of NGF-based approach in neurological disorders.

Disruption of redox homeostasis for combinatorial drug efficacy in K-Ras tumors as revealed by metabolic connectivity profiling.

BACKGROUND: Rewiring of metabolism induced by oncogenic K-Ras in cancer cells involves both glucose and glutamine utilization sustaining enhanced, unrestricted growth. The development of effective anti-cancer treatments targeting metabolism may be facilitated by the identification and rational combinatorial targeting of metabolic pathways. METHODS: We performed mass spectrometric metabolomics analysis in vitro and in vivo experiments to evaluate the efficacy of drugs and identify metabolic connectivity. RESULTS: We show that K-Ras-mutant lung and colon cancer cells exhibit a distinct metabolic rewiring, the latter being more dependent on respiration. Combined treatment with the glutaminase inhibitor CB-839 and the PI3K/aldolase inhibitor NVP-BKM120 more consistently reduces cell growth of tumor xenografts. Maximal growth inhibition correlates with the disruption of redox homeostasis, involving loss of reduced glutathione regeneration, redox cofactors, and a decreased connectivity among metabolites primarily involved in nucleic acid metabolism. CONCLUSIONS: Our findings open the way to develop metabolic connectivity profiling as a tool for a selective strategy of combined drug repositioning in precision oncology.

The Spinal Extracellular Matrix Modulates a Multi-level Protein Net and Epigenetic Inducers Following Peripheral Nerve Injury.

The extracellular matrix (ECM) of the central nervous system (CNS) plays a pivotal role in the pathogenesis of several neurodegenerative and neuroinflammatory disorders. Among the major factors, matrix metalloproteinases (MMPs) are actively involved in ECM remodeling and directly affect neuro-glial interactions. Since disease-related functional alterations mostly rely on the proteome, modulation of MMPs activity may be a strategy to correct mechanisms behind neurological disorders. We here investigated modifications of signaling components related to the central pathways in spinal maladaptive plasticity following spared nerve injury (SNI) of the sciatic nerve, and after treatment with the MMPs inhibitor GM6001 for 3 or 8 days. We found that GM6001 reduced the massive astrocytic and microglial activation indicative of reactive gliosis. Functional activity of GM6001 was paralleled by its significant effect on expression levels of the purinergic P2X4 receptor (P2X4R), the transcription factors NFκB and RPBJ, as well as levels of the nerve growth factor (NGF) receptor TrkA. Moreover, we showed that histone deacetylases 1 and 2 (HDAC1, HDAC2) were differentially modulated after SNI and GM6001 treatments for 3 or 8 days. Our data suggest a multi-level network of interactions across ECM and the neuroglial network involving MMPs, the neurotrophin system, intracellular signaling, and epigenetic modifications.

Roadmap for Stroke: Challenging the Role of the Neuronal Extracellular Matrix.

Stroke is a major challenge in modern medicine and understanding the role of the neuronal extracellular matrix (NECM) in its pathophysiology is fundamental for promoting brain repair. Currently, stroke research is focused on the neurovascular unit (NVU). Impairment of the NVU leads to neuronal loss through post-ischemic and reperfusion injuries, as well as coagulatory and inflammatory processes. The ictal core is produced in a few minutes by the high metabolic demand of the central nervous system. Uncontrolled or prolonged inflammatory response is characterized by leukocyte infiltration of the injured site that is limited by astroglial reaction. The metabolic failure reshapes the NECM through matrix metalloproteinases (MMPs) and novel deposition of structural proteins continues within months of the acute event. These maladaptive reparative processes are responsible for the neurological clinical phenotype. In this review, we aim to provide a systems biology approach to stroke pathophysiology, relating the injury to the NVU with the pervasive metabolic failure, inflammatory response and modifications of the NECM. The available data will be used to build a protein-protein interaction (PPI) map starting with 38 proteins involved in stroke pathophysiology, taking into account the timeline of damage and the co-expression scores of their RNA patterns The application of the proposed network could lead to a more accurate design of translational experiments aiming at improving both the therapy and the rehabilitation processes.

ROS networks: designs, aging, Parkinson's disease and precision therapies.

How the network around ROS protects against oxidative stress and Parkinson's disease (PD), and how processes at the minutes timescale cause disease and aging after decades, remains enigmatic. Challenging whether the ROS network is as complex as it seems, we built a fairly comprehensive version thereof which we disentangled into a hierarchy of only five simpler subnetworks each delivering one type of robustness. The comprehensive dynamic model described in vitro data sets from two independent laboratories. Notwithstanding its five-fold robustness, it exhibited a relatively sudden breakdown, after some 80 years of virtually steady performance: it predicted aging. PD-related conditions such as lack of DJ-1 protein or increased α-synuclein accelerated the collapse, while antioxidants or caffeine retarded it. Introducing a new concept (aging-time-control coefficient), we found that as many as 25 out of 57 molecular processes controlled aging. We identified new targets for "life-extending interventions": mitochondrial synthesis, KEAP1 degradation, and p62 metabolism.

Neurons, Glia, Extracellular Matrix and Neurovascular Unit: A Systems Biology Approach to the Complexity of Synaptic Plasticity in Health and Disease.

The synaptic cleft has been vastly investigated in the last decades, leading to a novel and fascinating model of the functional and structural modifications linked to synaptic transmission and brain processing. The classic neurocentric model encompassing the neuronal pre- and post-synaptic terminals partly explains the fine-tuned plastic modifications under both pathological and physiological circumstances. Recent experimental evidence has incontrovertibly added oligodendrocytes, astrocytes, and microglia as pivotal elements for synapse formation and remodeling (tripartite synapse) in both the developing and adult brain. Moreover, synaptic plasticity and its pathological counterpart (maladaptive plasticity) have shown a deep connection with other molecular elements of the extracellular matrix (ECM), once considered as a mere extracellular structural scaffold altogether with the cellular glue (i.e., glia). The ECM adds another level of complexity to the modern model of the synapse, particularly, for the long-term plasticity and circuit maintenance. This model, called tetrapartite synapse, can be further implemented by including the neurovascular unit (NVU) and the immune system. Although they were considered so far as tightly separated from the central nervous system (CNS) plasticity, at least in physiological conditions, recent evidence endorsed these elements as structural and paramount actors in synaptic plasticity. This scenario is, as far as speculations and evidence have shown, a consistent model for both adaptive and maladaptive plasticity. However, a comprehensive understanding of brain processes and circuitry complexity is still lacking. Here we propose that a better interpretation of the CNS complexity can be granted by a systems biology approach through the construction of predictive molecular models that enable to enlighten the regulatory logic of the complex molecular networks underlying brain function in health and disease, thus opening the way to more effective treatments.

Differential Modulation of NF-κB in Neurons and Astrocytes Underlies Neuroprotection and Antigliosis Activity of Natural Antioxidant Molecules.

Neuroinflammation, a hallmark of chronic neurodegenerative disorders, is characterized by sustained glial activation and the generation of an inflammatory loop, through the release of cytokines and other neurotoxic mediators that cause oxidative stress and limit functional repair of brain parenchyma. Dietary antioxidants may protect against neurodegenerative diseases by counteracting chronic neuroinflammation and reducing oxidative stress. Here, we describe the effects of a number of natural antioxidants (polyphenols, carotenoids, and thiolic molecules) in rescuing astrocytic function and neuronal viability following glial activation by reducing astrocyte proliferation and restoring astrocytic and neuronal survival and basal levels of reactive oxygen species (ROS). All antioxidant molecules are also effective under conditions of oxidative stress and glutamate toxicity, two maladaptive components of neuroinflammatory processes. Moreover, it is remarkable that their antioxidant and anti-inflammatory activity occurs through differential modulation of NF-κB binding activity in neurons and astrocytes. In fact, we show that inflammatory stimuli promote a significant induction of NF-κB binding activity in astrocytes and its concomitant reduction in neurons. These changes are prevented in astrocytes and neurons pretreated with the antioxidant molecules, suggesting that NF-κB plays a key role in the modulation of survival and anti-inflammatory responses. Finally, we newly demonstrate that effective antigliosis and neuroprotective activity is achieved with a defined cocktail of four natural antioxidants at very low concentrations, suggesting a promising strategy to reduce inflammatory and oxidative damage in neurodegenerative diseases with limited side effects.

Neural plasticity and adult neurogenesis: the deep biology perspective.

The recognition that neurogenesis does not stop with adolescence has spun off research towards the reduction of brain disorders by enhancing brain regeneration. Adult neurogenesis is one of the tougher problems of developmental biology as it requires the generation of complex intracellular and pericellular anatomies, amidst the danger of neuroinflammation. We here review how a multitude of regulatory pathways optimized for early neurogenesis has to be revamped into a new choreography of time dependencies. Distinct pathways need to be regulated, ranging from neural growth factor induced differentiation to mitochondrial bioenergetics, reactive oxygen metabolism, and apoptosis. Requiring much Gibbs energy consumption, brain depends on aerobic energy metabolism, hence on mitochondrial activity. Mitochondrial fission and fusion, movement and perhaps even mitoptosis, thereby come into play. All these network processes are interlinked and involve a plethora of molecules. We recommend a deep thinking approach to adult neurobiology.

Neuro-Immune Hemostasis: Homeostasis and Diseases in the Central Nervous System.

Coagulation and the immune system interact in several physiological and pathological conditions, including tissue repair, host defense, and homeostatic maintenance. This network plays a key role in diseases of the central nervous system (CNS) by involving several cells (CNS resident cells, platelets, endothelium, and leukocytes) and molecular pathways (protease activity, complement factors, platelet granule content). Endothelial damage prompts platelet activation and the coagulation cascade as the first physiological step to support the rescue of damaged tissues, a flawed rescuing system ultimately producing neuroinflammation. Leukocytes, platelets, and endothelial cells are sensitive to the damage and indeed can release or respond to chemokines and cytokines (platelet factor 4, CXCL4, TNF, interleukins), and growth factors (including platelet-derived growth factor, vascular endothelial growth factor, and brain-derived neurotrophic factor) with platelet activation, change in capillary permeability, migration or differentiation of leukocytes. Thrombin, plasmin, activated complement factors and matrix metalloproteinase-1 (MMP-1), furthermore, activate intracellular transduction through complement or protease-activated receptors. Impairment of the neuro-immune hemostasis network induces acute or chronic CNS pathologies related to the neurovascular unit, either directly or by the systemic activation of its main steps. Neurons, glial cells (astrocytes and microglia) and the extracellular matrix play a crucial function in a "tetrapartite" synaptic model. Taking into account the neurovascular unit, in this review we thoroughly analyzed the influence of neuro-immune hemostasis on these five elements acting as a functional unit ("pentapartite" synapse) in the adaptive and maladaptive plasticity and discuss the relevance of these events in inflammatory, cerebrovascular, Alzheimer, neoplastic and psychiatric diseases. Finally, based on the solid reviewed data, we hypothesize a model of neuro-immune hemostatic network based on protein-protein interactions. In addition, we propose that, to better understand and favor the maintenance of adaptive plasticity, it would be useful to construct predictive molecular models, able to enlighten the regulating logic of the complex molecular network, which belongs to different cellular domains. A modeling approach would help to define how nodes of the network interact with basic cellular functions, such as mitochondrial metabolism, autophagy or apoptosis. It is expected that dynamic systems biology models might help to elucidate the fine structure of molecular events generated by blood coagulation and neuro-immune responses in several CNS diseases, thereby opening the way to more effective treatments.

Differentiation by nerve growth factor (NGF) involves mechanisms of crosstalk between energy homeostasis and mitochondrial remodeling.

Neuronal differentiation involves extensive modification of biochemical and morphological properties to meet novel functional requirements. Reorganization of the mitochondrial network to match the higher energy demand plays a pivotal role in this process. Mechanisms of neuronal differentiation in response to nerve growth factor (NGF) have been largely characterized in terms of signaling, however, little is known about its impact on mitochondrial remodeling and metabolic function. In this work, we show that NGF-induced differentiation requires the activation of autophagy mediated by Atg9b and Ambra1, as it is disrupted by their genetic knockdown and by autophagy blockers. NGF differentiation involves the induction of P-AMPK and P-CaMK, and is prevented by their pharmacological inhibition. These molecular events correlate with modifications of energy and redox homeostasis, as determined by ATP and NADPH changes, higher oxygen consumption (OCR) and ROS production. Our data indicate that autophagy aims to clear out exhausted mitochondria, as determined by enhanced localization of p62 and Lysotracker-red to mitochondria. In addition, we newly demonstrate that NGF differentiation is accompanied by increased mitochondrial remodeling involving higher levels of fission (P-Drp1) and fusion proteins (Opa1 and Mfn2), as well as induction of Sirt3 and the transcription factors mtTFA and PPARγ, which regulate mitochondria biogenesis and metabolism to sustain increased mitochondrial mass, potential, and bioenergetics. Overall, our data indicate a new NGF-dependent mechanism involving mitophagy and extensive mitochondrial remodeling, which plays a key role in both neurogenesis and nerve regeneration.

Divergent in vitro/in vivo responses to drug treatments of highly aggressive NIH-Ras cancer cells: a PET imaging and metabolomics-mass-spectrometry study.

Oncogenic K-ras is capable to control tumor growth and progression by rewiring cancer metabolism. In vitro NIH-Ras cells convert glucose to lactate and use glutamine to sustain anabolic processes, but their in vivo environmental adaptation and multiple metabolic pathways activation ability is poorly understood. Here, we show that NIH-Ras cancer cells and tumors are able to coordinate nutrient utilization to support aggressive cell proliferation and survival. Using PET imaging and metabolomics-mass spectrometry, we identified the activation of multiple metabolic pathways such as: glycolysis, autophagy recycling mechanism, glutamine and serine/glycine metabolism, both under physiological and under stress conditions. Finally, differential responses between in vitro and in vivo systems emphasize the advantageous and uncontrolled nature of the in vivo environment, which has a pivotal role in controlling the responses to therapy.

Rotenone down-regulates HSPA8/hsc70 chaperone protein in vitro: A new possible toxic mechanism contributing to Parkinson's disease.

HSPA8/hsc70 (70-kDa heat shock cognate) chaperone protein exerts multiple protective roles. Beside its ability to confer to the cells a generic resistance against several metabolic stresses, it is also involved in at least two critical processes whose activity is essential in preventing Parkinson's disease (PD) pathology. Actually, hsc70 protein acts as the main carrier of chaperone-mediated autophagy (CMA), a selective catabolic pathway for alpha-synuclein, the main pathogenic protein that accumulates in degenerating dopaminergic neurons in PD. Furthermore, hsc70 efficiently fragments alpha-synuclein fibrils in vitro and promotes depolymerization into non-toxic alpha-synuclein monomers. Considering that the mitochondrial complex I inhibitor rotenone, used to generate PD animal models, induces alpha-synuclein aggregation, this study was designed in order to verify whether rotenone exposure leads to hsc70 alteration possibly contributing to alpha-synuclein aggregation. To this aim, human SH-SY5Y neuroblastoma cells were treated with rotenone and hsc70 mRNA and protein expression were assessed; the effect of rotenone on hsc70 was compared with that exerted by hydrogen peroxide, a generic oxidative stress donor with no inhibitory activity on mitochondrial complex I. Furthermore, the effect of rotenone on hsc70 was verified in primary mouse cortical neurons. The possible contribution of macroautophagy to rotenone-induced hsc70 modulation was explored and the influence of hsc70 gene silencing on neurotoxicity was assessed. We demonstrated that rotenone, but not hydrogen peroxide, induced a significant reduction of hsc70 mRNA and protein expression. We also observed that the toxic effect of rotenone on alpha-synuclein levels was amplified when macroautophagy was inhibited, although rotenone-induced hsc70 reduction was independent from macroautophagy. Finally, we demonstrated that hsc70 gene silencing up-regulated alpha-synuclein mRNA and protein levels without affecting cell viability and without altering rotenone- and hydrogen peroxide-induced cytotoxicity. These findings demonstrate the existence of a novel mechanism of rotenone toxicity mediated by hsc70 and indicate that dysfunction of both CMA and macroautophagy can synergistically exacerbate alpha-synuclein toxicity, suggesting that hsc70 up-regulation may represent a valuable therapeutic strategy for PD.

Modulation of Matrix Metalloproteinases Activity in the Ventral Horn of the Spinal Cord Re-stores Neuroglial Synaptic Homeostasis and Neurotrophic Support following Peripheral Nerve Injury.

Modulation of extracellular matrix (ECM) remodeling after peripheral nerve injury (PNI) could represent a valid therapeutic strategy to prevent maladaptive synaptic plasticity in central nervous system (CNS). Inhibition of matrix metalloproteinases (MMPs) and maintaining a neurotrophic support could represent two approaches to prevent or reduce the maladaptive plastic changes in the ventral horn of spinal cord following PNI. The purpose of our study was to analyze changes in the ventral horn produced by gliopathy determined by the suffering of motor neurons following spared nerve injury (SNI) of the sciatic nerve and how the intrathecal (i.t.) administration of GM6001 (a MMPs inhibitor) or the NGF mimetic peptide BB14 modulate these events. Immunohistochemical analysis of spinal cord sections revealed that motor neuron disease following SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) response in the ventral horn of the spinal cord, indicative of reactive gliosis. These changes were paralleled by decreased glial aminoacid transporters (glutamate GLT1 and glycine GlyT1), increased levels of the neuronal glutamate transporter EAAC1, and a net increase of the Glutamate/GABA ratio, as measured by HPLC analysis. These molecular changes correlated to a significant reduction of mature NGF levels in the ventral horn. Continuous i.t. infusion of both GM6001 and BB14 reduced reactive astrogliosis, recovered the expression of neuronal and glial transporters, lowering the Glutamate/GABA ratio. Inhibition of MMPs by GM6001 significantly increased mature NGF levels, but it was absolutely ineffective in modifying the reactivity of microglia cells. Therefore, MMPs inhibition, although supplies neurotrophic support to ECM components and restores neuro-glial transporters expression, differently modulates astrocytic and microglial response after PNI.